The treatmant of patients with glioblastoma and pancreatic carcinoma remains a challenge. In the present study, we found that pretreatment with the novel Hsp90 inhibitor NVP-HSP990 strongly sensitizes U251 glioma and MIA PaCa-2 pancreatic carcinoma cells to hyperthermia and IR and particulary to the combination thereof. The triple combination caused a significant reduction in cell number which was associated with a morphological alterations typical of mitotic catastrophe and apoptosis.
There is a lot of experimental [16–18] and clinical evidence that hyperthermia can increase the effectiveness of other conventional treatments such chemotherapy  or especially radiotherapy [20, 21]. The exact mechanism how hyperthermia increases radiosensitivity is still not completely elucidated but it has been proposed that hyperthermia may interfere with radiation-induced DNA damage . On the other hand, heat shock proteins mediate resistance to hyperthermia . It has been shown that inhibition of Hsp90 with geldanamycin causes delayed and impaired recovery from heat shock . In HEK293 cells, specific inhibition of Hsp90 together with short term exposure (20–60 min) to 42°C was highly cytotoxic, through accelerated degradation of Cdc25A , a member of the CDC25 family of phosphatases which is specifically degraded in response to DNA damage . These findings support the hypothesis that the antineoplastic effect of hyperthermia could be potentiated by concurrent inhibition of Hsp90. In our experimental conditions, we observed that U251 and MIA PaCa-2 cells which were incubated for 1 hour at 42°C and concurrently treated with NVP-HSP990 showed a significantly lower capability to form colonies in comparison to cells which were treated with only one of the two modalities.
The effects of combined hyperthermia and irradiation treatment depend on many different factors such a heating temperature, heating time, sequence and time interval between the two modalities . Despite intensive research, it is still not clear wheather hyperthermia before or after irradiation causes a more pronounced enhancement of radiation damage. Probably, this effect is cell-type specific. In the case of concurrent irradiation and hyperthermia, maximal additive/synergistic effects can be expected while increasing the intervals of time between hyperthermia and IR, regardless of sequence, will abrogate the radiosensitisation induced by hyperthermia . As expected, because of the time interval of 23 hours between hyperthermia and IR, we observed only a weak influence of hyperthermia on radiosensitivity, cell cycle distribution, induction of mitotic catastrophe and apoptosis.
It has been proposed that the radiosensitising effect of Hsp90 inhibitors is caused by degradation of several proteins such a ErbB2, EGFR, Raf-1 and Akt [29, 30] which reportedly can influence radioresistance. DNA repair and cell cycle checkpoint activation are other proposed mechanisms by which Hsp90 can influence the DNA damage response to IR . A previous study reported that a 24 h-pretreatment with an Hsp 90 inhibitor similar to NVP-HSP990, NVP-BEP800, caused an increase in radiosensitivity in two glioblastoma, one lung carcinoma and one fibrosarcoma cell line through cell-cycle impairment, increased DNA damage and repair protraction . The authors found that changes in the expression of survival markers (Hsp90, Hsp70, Akt, phospho-Akt, Raf-1 and survivin), an apoptosis-associated protein (cleaved caspase 3) or of the functional p53 status did not significantly contribute to the sensitivity of two out of four tested cell lines to NVP-BEP800 alone or in combination with IR. Another group found that the geldanamycin-based Hsp90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) enhances radiosensitization of human U251 and MIA PaCa-2 cells . The treatment of the cells with this compound caused a reduction of the expression of Akt, Raf-1 and especially ErbB2. The authors further reported that treatment of DU145 prostate carcinoma cells with 17-DMAG abrogated the G2- and S-phase cell cycle checkpoints and enhanced the radiosensitivity of the cells.
It has been reported that treatment of the human lung adenocarcinoma cell line A549 with KNK437, an benzylidene lactam compound which acts as a heat shock protein inhibitor, causes the enhancement of thermal radiosensitization in mild hyperthermia combined with low dose IR . In the same study, it has been demonstrated that KNK437 caused inhibition of Hsp72 and Hsp27 expression. NVP-HSP990 shows a different mechanism of action; it binds to the NH2-terminal ATP-binding pocket of Hsp90 while KNK437 inhibits synthesis of various heat shock proteins at the mRNA level. KNK437 has also been proposed to induce radioresistance of A-172 human glioblastoma and human squamous cell carcinoma cells .
In our experiments, we observed that pretreatment of U251 cells with NVP-HSP990 and hyperthermia before irradiation with 4 Gy caused a delayed acumulation of cells in the G2/M phase. The strongest effect was detected 24 hours after irradiation. Three to five days after the irradiation teatment, we observed a strong increase of the number of cells with morphological signs of mitotic catastrophe (micro- and multinucleated cells) [34, 35]. The number of apoptotic cells also increased. As large numbers of necrotic cells (taking up PI) were not found, this suggests that mitotic catastrophe constitutes a prelude to apoptotic cell death. Similar findings were reported for a novel small molecule inhibitor that lowers the threshold for Hsf1 (Heat shock factor protein 1) activation . The inhibition enhanced thermal sensitivity and significant thermal radiosensitization followed by loss of mitochondrial potential and mitotic catastrophe in HT29 colon carcinoma cells.